不同剂量雷公藤多苷对寻常型银屑病样小鼠血清炎性因子的影响

Effects of different doses of tripterygium glycosides on serum inflammatory factors in psoriatic vulgaris-like mice

目的观察不同剂量雷公藤多苷对寻常型银屑病样小鼠血清炎性因子白细胞介素-6(IL-6)、白细胞介素-17(IL-17)、白细胞介素-23(IL-23)、肿瘤坏死因子-α(TNF-α)和核转录因子-κB(NF-κB)的影响,并探讨其最佳治疗剂量。方法2019年2—11月,将40只BALB/c雄性小鼠采用随机数字表法分为五组(n=8):对照组、模型组,低、中、高剂量组给药组。小鼠建模后给药组采用不同剂量雷公藤多苷灌胃,浓度为10 mg·kg^(-1)·d^(-1)、20 mg·kg^(-1)·d^(-1)和40 mg·kg^(-1)·d^(-1),持续灌胃14 d。收集小鼠病变皮肤组织,对超氧化物歧化酶(SOD)活性及谷胱甘肽(GSH)和丙二醛(MDA)水平进行测定。酶联免疫吸附测定(ELISA)法检测各组小鼠血清IL-6、IL-17、IL-23和TNF-α水平,Western blotting法检测小鼠血清NF-κB水平。细胞处理后流式细胞术检测各组细胞周期。结果病变皮肤均质化后SOD活性、GSH和MDA水平测定结果显示,模型组小鼠的GSH水平(8.94±0.95)U/mgprot和SOD活性(21.37±3.76)U/mgprot低于对照组(30.93±1.91)U/mgprot、(44.35±4.09)U/mgprot,(P=0.002、0.007),给药组小鼠的GSH水平和SOD活性高于模型组,其中中剂量组最高,为(36.03±1.34)U/mgprot、(42.45±2.43)U/mgprot,P<0.001、P=0.003,低剂量组最低为(26.86±2.03)U/mgprot、(30.39±2.04)U/mgprot,均P<0.05;而MDA测定结果相反,模型组小鼠MDA水平高于对照组,(74.64±2.29)nmol/mgprot比(40.51±2.34)nmol/mgprot,P<0.001,经过药物治疗后降低(P<0.001)。ELISA结果显示,与对照组相比,模型组小鼠血清中IL-6、IL-17、IL-23和TNF-α表达水平升高为(4.93±0.09)pg/mg、(9.05±0.20)pg/mg、(5.23±0.17)pg/mg、(11.51±1.23)pg/mg,均P<0.05;低剂量组小鼠IL-17和TNF-α的表达水平与模型组相比差异无统计学意义(均P>0.05),中剂量组(2.76±0.23)pg/mg、(6.12±0.27)pg/mg、(3.19±0.21)pg/mg、(8.21±0.44)pg/mg和高剂量组(3.72±0.09)pg/mg、(6.02±0.25)pg/mg、(3.37±0.41)pg/mg、(8.44±0.38)pg/mg小鼠血清炎性因子下降,均P<0.05。Western blotting结果显示,与对照组相比,模型组小鼠的NF-κB表达水平升高(P<0.001),给药组小鼠NF-κB表达水平降低(P=0.005),且高剂量组小鼠的抑制效果最佳(P<0.001)。流式细胞术结果显示,与对照组相比,10 mg/L雷公藤多苷处理48 h的HaCat细胞的G1期细胞所占比例差异无统计学意义(P=0.300),而20 mg/L和40 mg/L雷公藤多苷处理48 h后细胞G1期细胞所占比例升高(P<0.001,P=0.013)。结论雷公藤多苷可以降低银屑病样小鼠血清炎性因子,抑制NF-κB表达,调节病变皮肤抗氧化水平,并且最佳使用量在20 mg·kg^(-1)·d^(-1)左右。

Objective To observe the effects of different doses of tripterygium glycosides on serum inflammatory factors IL-6,IL-17,IL-23,TNF-αand NF-κB in psoriasis vulgaris mice,and to explore the optimal therapeutic dose.Methods From February 2019 to November 2019,40 male BALB/c mice were randomly assigned into 5 groups(n=8)using a random number table method:the blank control group,the model control group,and the low,medium,and high dose groups,respectively.After modelling,the drug-administered group was administered with different doses of tripterygium glycosides at a concentration of 10 mg·kg^(-1)·d^(-1),20 mg·kg^(-1)·d^(-1) and 40 mg·kg^(-1)·d^(-1) for 14 days.Diseased skin tissues of mice were collected,and SOD activity,GSH and MDA levels were measured.The serum levels of IL-6,IL-17,IL-23 and TNF-αin each group of mice were detected by ELISA,and the serum levels of NF-κB were analyzed by Western blotting.After cell treatment,the cell cycle of each group was detected by flow cytometry.Results After homogenization of the diseased skin,the determination result of SOD activity and GSH and MDA levels showed that the GSH[(8.94±0.95)U/mgprot vs.(30.93±1.91)U/mgprot]level and SOD[(21.37±3.76)U/mgprot vs.(44.35±4.09)U/mgprot]activity of mice in model group were significantly lower than those in control group(all P<0.05);GSH and SOD in the drug-administered groups were significantly higher than those in the model group,of which the middle-dose group was the highest[GSH:(36.03±1.34)U/mgprot,SOD:(42.45±2.43)U/mgprot,all P<0.05]and the low dose group was the lowest[(26.86±2.03)U/mgprot,(30.39±2.04)U/mgprot,all P<0.05].However,the results of MDA were reversed.The MDA in the model group[(74.64±2.29)nmol/mgprot]was higher than that in the control group[(40.51±2.34)nmol/mgprot,P<0.001],and it was significantly reduced after drug treatment(P<0.001).The results of ELISA showed that expression levels of IL-6(4.93±0.09)pg/mg,IL-17(9.05±0.20)pg/mg,IL-23(5.23±0.17)pg/mg and TNF-α(11.51±1.23)pg/mg in the model group were significantly higher than those in the control group(all P<0.05).There was no significant difference in the expression levels of IL-17 and TNF-αbetween the the low dose group and the model group(all P>0.05),and the serum inflammatory factors(IL-6,IL-17,IL-23 and TNF-α)in mice of the medium dose group[(2.76±0.23)pg/mg,(6.12±0.27)pg/mg,(3.19±0.21)pg/mg,(8.21±0.44)pg/mg]and the high dose group[(3.72±0.09)pg/mg,(6.02±0.25)pg/mg,(3.37±0.41)pg/mg,(8.44±0.38)pg/mg]decreased significantly compared with the model group(all P<0.05).The western blotting results showed that the expression level of NF-κB significantly increased in model group(P<0.001)and significantly reduced in the drug-administered groups compared with control group(P=0.005),and the inhibitory effect in high dose group was the best(P<0.001).In the flow cytometry results,compared with the control group,there was no significant difference in the proportion of G1 phase cells of HaCat cells treated with 10μg/ml triptolide for 48 h(P=0.3).After treatment with 20 mg/L and 40 mg/L tripterygium glycosides for 48 h,the proportion of cells in G1 phase increased significantly(P<0.001,P=0.013).Conclusion Tripterygium glycosides can reduce serum inflammatory factors,inhibit NF-κB expression,and regulate the level of antioxidant skin disease in psoriasis-like mice.The optimal dosage is about 20 mg·kg^(-1)·d^(-1).